Spontaneous precipitation reaction is that the mixture of calcium and supersaturation solution of carbonate are simply blended. It is the most important and simplest method to prepare CCNM (Sun et al., 2020). Persano et al. (2022) synthesized CCNM using this method and studied in detail the effects of different synthesis parameters. They found that as the stirring speed increased, the nano size of CaCO₃ decreased correspondingly. Meanwhile, it was found that the higher the salt concentration, the higher the supersaturation became, and thus the smaller the nano size. Yang et al. (2021a) used CaCl₂ and NaCO₃ as raw materials, injected NaCO₃ into the CaCl₂ containing Ca(OH)₂ to prepare CCNM, and studied the use of Ca(OH)₂ as an additive. As a non-impurity additive, Ca(OH)₂ increased the pH value of the suspension. The results showed that the higher the pH value of the reaction system, the smaller the CCNM size. Pérez-Villarejo et al. (2018) reported a simple and rapid method for synthesizing CCNM. The experiment used Ca(NO₃)₂·4H₂O and NaHCO₃ as raw materials and mainly observed the role of sucrose as an additive in the synthesis of CCNM. Their conclusion is that sucrose interacts with Ca²⁺ and water molecules through hydroxyl groups. The higher the concentration of sucrose, the more crystalline forms of synthetic vaterite, and the smaller the size of nanoparticles. Sucrose can stabilize the vaterite crystal form, making it difficult to transform into a more stable calcite crystal form, which provides a fast and simple method for the synthesis of vaterite crystal form in the future.

Based on the application experience, the precipitation method has been reported to involve the addition of different molecules, including synthetic polymers, surfactants and biomolecules, to synthesize CCNM of different sizes and shapes. This method can adjust the crystal morphology and particle size of CCNM by changing the concentration of reactants and adding organic matter. However, the rapid reaction speed of precipitation reaction, which is not conducive to observing and studying the reaction process. This might be its main drawback.

The mechano-chemical synthesis method to produce CCNM is the process involving mechanical fragmentation and chemical reactions (Cestari et al., 2021). Its chemical reaction occurs through the absorption of mechanical energy by reactants. This method is divided into the dry mechano-chemical and the wet mechano-chemical method (Mkhize et al., 2021). Gbadenyan et al. (2021) prepared CCNM by mechano-chemical grinding method. They started with using stainless steel balls and cans to grind the snail shell to obtain fine particles. Then, the fine particles are placed in a stainless steel wide-mouthed bottle, solvent is added, and wet grinding is performed to obtain CCNM. Compared with precipitation method, the equipment setup for the mechano-chemical method is cost-effective and enables the agglomeration-free production of nanoparticles with narrow particle size distribution (Piras et al., 2019). But this method also has some drawbacks, such as instrument rusting, which may hinder the synthesis process of nanoparticles and cause certain pollution.

It is advantageous to synthesize CCNM using natural reserves of CaCO₃, such as eggshells and seashells, as it is easy to obtain, non-toxic, and biocompatible, making it an ideal candidate for biomedical applications (Prihanto et al., 2023). Rinu et al. (2020) synthesized nanoparticles using eggshells as raw materials. By collecting a large amount of eggshells and drying them at room temperature. Then gently crush the eggshell and place it in a crucible, keeping it in a muffle furnace at 900°C for 2 h. Finally, it is turned into fine powder to synthesize CCNM. Khan et al. (2019) used eggshells and agar as raw materials to calcine the synthesized products at high temperatures, ultimately synthesizing CCNM. The calcination temperature has a significant impact on the morphology, composition, and size of particles. Hussein et al. (2020) crushed the shells into powder and mixed them with HCl to form CaCl₂. And the effect of different volumes of double-distilled water (DDW) on the morphology of CCNM derived from shells was studied. Nanoparticles prepared with different volumes of DDW have different shapes and sizes, which is due to the dilution of the solution volume by DDW, resulting in changes in the activity space of the nanoparticles.

Seashells and eggshells are abundant in resources, inexpensive, and easy to obtain, which can reduce environmental pollution levels and have good economic and social benefits. From this, it can be seen that CCNM obtained through shells and eggshells has enormous advantages, but the resulting nanoparticles are impure, which requires further exploration.

The gas diffusion method has been widely used to synthesize CCNM in the biomedical field. This method involves the thermal decomposition of (NH₄)₂CO₃ or NH₄HCO₃ to generate CO₂ and NH₃ diffusion into an ethanol solution containing calcium salts to generate precipitates or the introduction of CO₂ gas into the calcium salt ethanol solution at atmospheric pressure to synthesize CCNM (Wang et al., 2020). Chuzeville et al. (2022) used this method to synthesize the dispersed CCNM below 150 nm in ethanol. Ethanol is the main solvent to ultimately obtain stable and uniform spherical nanoparticles, which can inhibit the spontaneous aggregation and crystallization of CCNM. Ju et al. (2022) made some improvements on the basis of the original steps. In the ethanol-water binary system, stable amorphous calcium carbonate (ACC) nanospheres were synthesized by gas diffusion method. And different volumes of ammonia were added to the solution, it was observed that as ammonia was continuously added, the volume of the nanomaterials continued to decrease. Xu et al. (2022a) synthesized ACC by gas diffusion method, added Ca(OH)₂ and CO₂ in the methanol-water system, reacted in an autoclave and centrifuged to obtain ACC. The research results indicate that with the increase of water content in the solution system, ACC gradually changed into a metastable vaterite crystal.

Compared to other synthesis methods, gas-phase diffusion method has the advantage of controllable reaction speed. There is no need for other additives, and higher product quality could be acquired. Thus, it is often used as the preferred method for studying the biomimetic synthesis of CCNM minerals. But the obtained nanoparticles are still easy to agglomerate. Polyethylene glycol and other substances can be used to modify the surface of CCNM to improve the dispersion and stability in aqueous solution.

An ideal anticancer drug delivery system is to deliver the drug directly to the target site, with minimal impact on normal cells, thereby improving treatment efficiency and producing minimal toxic side effects. Numerous tumor targeted drug delivery systems have been constructed through nanotechnology, which not only improves drug stability but also limits drug toxicity (Elbaz et al., 2020). CCNM have received great attention among different inorganic nanocarriers. Their natural characteristics such as biocompatibility, pH responsiveness and high encapsulation efficiency make them an ideal carrier for transporting various bioactive substances, especially anticancer drugs and genes (Fu et al., 2019). Therefore, when CCNM are coupled with drugs and genes, they improve the efficiency of disease treatment.

At present, there are two main ways for CCNM to deliver drugs for tumor treatment. One method involves directly binding anti-tumor drugs onto the surface of CCNM, while the other method involves co-doping drugs with CCNM and allowing the complex to enter tumor cells (Teixeira et al., 2022). Zheng et al. (2022) used the one-pot gas diffusion method to co-dope curcumin (CUR) with CCNM, and the obtained CCNM can decompose at low pH to release CUR. Among them, CUR causes cell apoptosis by affecting Ca²⁺ homeostasis. By using CCNM as a carrier to transport CUR, the programmed release of CUR at the tumor site was achieved, improving the transport efficiency and anticancer activity of CUR.

Popova et al. (2021) loaded DOX onto the surface of CCNM, with the aim of studying the coupling efficiency of CCNM with drugs and the effectiveness of carrying DOX in inhibiting cancer cell proliferation in vitro. In an in vitro model, the composite material of CCNM and DOX was shown to effectively inhibit the growth of cancer cells. The characteristics of CCNM also help prevent unnecessary accumulation of DOX in major organs such as the liver, heart, and kidneys.

On this basis, drug delivery systems can be designed to combine multiple treatment methods into an intelligent carrier, providing a solution for their potential applications in cancer diagnosis and treatment. As shown in Figure 3A, Yu et al. (2023) co-doped CCNM with indocyanine green (IGG) before loading DOX on the surface, then encapsulated them with poly (lactic-co-glycolic acid)-ss-chondroitin sulfate A (PSC). This study combines photothermal therapy (PTT) with chemotherapy to treat tumors. PTT can use photosensitizers to generate heat energy and ultimately eliminate tumor cells. IGG is an effective photosensitizer, but free IGG cannot selectively reach the tumor site. Utilizing CCNM delivered IGG to reach the tumor site for PTT. This method not only enables IGG to accurately reach tumor cells but also significantly increases the metabolic time of IGG and improves treatment efficiency. As shown in Figure 3B, compared with other control groups, the experimental group significantly inhibited the tumor volume of mice. It was observed in the mouse thermal image in Figure 3C that this nanomaterial helps IGG to better exert its therapeutic effect. Observing from the picture, the tumor temperature in the 5% glucose group was slightly increased after 5 min of laser irradiation. The free DOX + ICG group exhibited the tumor temperature with an increase to 45.4°C. Notably, PSC/ICG@ and PSC/ICG@+DOX groups showed an increased temperature of 60.5°C after 5 min irradiation, which could effectively generate hyperthermia for PTT of malignant tumors. From this, it can be seen that CCNM have enormous advantages in delivery systems. At the same time, we can further study its synthesis method and functionalization to generate CCNM with different morphologies, thereby achieving higher loading efficiency and achieving better therapeutic effects.

As a pivotal second messenger in cellular signaling, Ca²⁺ orchestrates the precise regulation of diverse physiological processes through targeted activation of specific proteins (Liu et al., 2020c). Under normal circumstances, the concentration of cytoplasmic free Ca²⁺ is maintained at a significantly low level of approximately 100 nmol/L. There is a 10⁴ ∼ 10⁵ -fold gradient of Ca²⁺ concentration between outside and inside of cells (Bai et al., 2022). Without the stimulation of abnormal conditions, the intracellular Ca²⁺ concentration is lower than the level of extracellular fluid, which plays an active role in regulating cells (Ma et al., 2020; Eisner et al., 2023). But if the concentration of Ca²⁺ exceeds the normal range, it will cause damage to the cells. Therefore, regulating Ca²⁺ concentration has broad research prospects in tumor treatment. In recent years, Ca²⁺ overload treatment has garnered significant attention in the field of tumor treatment, with a close association between Ca²⁺ and mitochondria (Zhu et al., 2019). When intracellular Ca²⁺ overload occurs, the mitochondrial Ca²⁺ uniporter (MCU) sequesters intracellular Ca²⁺. Upon reaching a certain threshold, this uptake of Ca²⁺ by mitochondria triggers the opening of the mitochondrial permeability transition pore (PTP) (Glitsch, 2019; Delierneux et al., 2020). When PTP is opened, many large molecules non-selectively diffuse from cytoplasm to mitochondria, leading to the destruction and dysfunction of mitochondrial membrane potential. Mitochondrial Ca²⁺ overload can lead to the release of cytochrome C and the activation of aspartate proteolytic enzymes, thereby damaging the production of reactive oxygen species related to the respiratory chain and affecting mitochondrial function, ultimately leading to cell apoptosis (Calvo-Rodriguez and Bacskai, 2021). After mitochondrial damage, biological transmission electron microscopy (Bio-TEM) is commonly employed to examine the morphology and abundance of mitochondria. At the same time, Flou-4 AM calcium probe is commonly used to detect intracellular Ca²⁺ (Zheng et al., 2021), while the Rhod-2 AM probe is used for detecting Ca²⁺ in mitochondria (Wang et al., 2023b).

Inadequate Ca²⁺ at the tumor site, intracellular Ca²⁺ channels can also effectively regulate the concentration of Ca²⁺, so it is difficult to achieve effective Ca²⁺ overload. Providing Ca²⁺ solely through calcium materials cannot achieve satisfactory results (Kong et al., 2021). Therefore, more and more collaborative treatment methods have been developed and have attracted widespread attention. For example, combining calcium nanomaterials with treatment methods such as chemotherapy can not only provide Ca²⁺, but also disrupt calcium homeostasis, thereby achieving more effective Ca²⁺ overload. As shown in Figure 4A, Li et al. (2021c) designed the M@CaCO₃@KAE nano platform, thus realizing the cooperative and targeted therapy of tumors. Among them, Kaempferol-3-O-Rutinoside (KAE) can effectively disrupt the regulation of Ca²⁺ homeostasis and promote Ca²⁺ influx, while CCNM can provide Ca²⁺, ultimately achieving Ca²⁺ overload and mitochondrial structure and function are disrupted, causing cytoskeletal collapse and oxidative stress, leading to cancer cell apoptosis. In Figures 4B, C, it was observed that the tumor volume of mice was effectively suppressed, indicating that this nanomaterial can to some extent inhibit tumor growth, which is of great significance for clinical applications. This experiment cleverly combines KAE with CaCO₃, greatly increasing the concentration of intracellular Ca²⁺, thereby achieving more efficient treatment of tumors with Ca²⁺ overload, providing a unique and novel approach for our future Ca²⁺ overload experiments. In addition, as shown in Figure 4D, Zheng et al. (2021) successfully prepared CCNM codoped with cisplatin (CDDP) and CUR. They destroyed the structure and function of mitochondria through CDDP, and destroyed the endoplasmic reticulum through CUR, so that a large amount of Ca²⁺ entered the cytoplasm, and CaCO₃ provided a large amount of Ca²⁺, thus achieving efficient Ca²⁺ overload. Due to the fluorescence characteristics of CUR, it was observed in Figure 4E that the fluorescence signal of the mouse tumor site became stronger within the first 12 h, further indicating that the CCNM nano platform has a certain degree of targeting and can accumulate within the tumor. The Ca²⁺ nanomodulator designed in this experiment achieves Ca²⁺ overload in multiple directions, targeting cancer through mitochondria, and has great prospects in clinical practice. Ca²⁺ overload is one of the most effective methods to induce cancer cell apoptosis. Upregulation of Ca²⁺ concentration in mitochondria can lead to a series of mitochondrial diseases, including decreased mitochondrial membrane potential, decreased ATP levels, altered mitochondrial morphology, and mitochondrial respiratory disorders (Guan et al., 2020). The use of ion interference to treat tumors is extremely ingenious in concept, providing a new perspective for tumor treatment. Ca²⁺ overload has gradually become a research hotspot, but simply killing tumor cells through Ca²⁺ overload is far from enough. We can use other methods for combined therapy to greatly improve the treatment efficiency of tumors.

Due to metabolic disorders in tumors, the extracellular pH of solid tumors is lower than that of normal tissues. The acidic extracellular environment of tumors enhances their invasiveness and metastasis, but there are few methods to selectively regulate the extracellular pH environment of tumors. It is impractical and non-selective to flush biological system instantaneously with alkaline liquid or proton pump inhibitor (Worsley et al., 2022). Among various methods for regulating the acidic environment of tumor cells, CCNM are particularly effective because they have efficient buffering ability and can directly regulate pH values.

On this basis, Som et al. (2016) prepare monodisperse CCNM with a size range of 20–300 nm, intentionally adjusting the pH value of the tumor microenvironment (TME). It utilized a mouse cancer model and repeated daily injections of CCNM, which significantly inhibited tumor growth. And in detecting dynamic pH values in tumor mice, it was observed that the pH value increased almost linearly during the first 30 min. This experiment demonstrates that CCNM can increase the pH value of tumors and inhibit tumor growth over time in tumors. It demonstrated for the first time the ability of using CCNM to regulate the pH value in solid tumors and observed that the effective alkalization of tumor acidic pH depends on the size of the nanoparticles. It should be noted that in order to achieve sustained tumor suppression effects in future experiments, it is necessary to optimize the dosage and use CCNM in combination with other therapies to improve the targeting of CCNM to tumors. Lam et al. (2021) co incubated cancer cells and fibroblasts and then treated them with CCNM. The results showed that nanoparticles selectively inhibited the growth of tumor cells, and reduced the cell migration of these cells, but had no effect on fibroblasts. This indicates that CCNM can limit the invasiveness of tumor cells without affecting the growth and behavior of surrounding stromal cells. The advantage of this experiment compared to other experiments is that it uses microfluidic devices to simulate the flow parameters in the body to determine the impact of pH changes on tumor survival and migration. Research has shown that CCNM can indeed alter the acidic microenvironment of tumors, but relying solely on CCNM to alter the acidic environment is still not enough.

To remedy the intrinsic deficits in energy production, cancer cells typically increase their uptake of extracellular glucose. Thus, the glucose deprivation is an effective way to cause the rapid and massive death of cancer cells, which is the essence of starving cancer cells. Based on the concept of cancer hunger treatment, as shown in Figure 5A, Li et al. (2020b) explored the effect of CCNM on coagulation. In Figure 5B, it was observed that in a mouse breast tumor model, CCNM were injected locally and physiological saline was used as the control. Through naked eye, HE, and Masson trichrome staining, it was found that CCNM, under the influence of acid stimulation, can cause coagulation reactions and thrombosis in tumor blood vessels, resulting in abnormal blood circulation and glucose supply. Therefore, CCNM have great prospects in the field of cancer treatment. Among them, Ca²⁺ activates prothrombin to thrombin, thereby causing blood to coagulate. And as the pH decreases, CCNM release more Ca²⁺, causing faster blood coagulation. The characteristic of CCNM that are pH sensitive and induce blood coagulation makes them a promising material for cancer hunger treatment. Although this study demonstrates that CCNM can induce coagulation in tumor blood vessels, it still cannot cover all tumor blood vessels, which requires further exploration.

In recent years, the development of high-performance contrast agents in magnetic resonance imaging (MRI) has received great attention. As shown in Figure 5C, Yi et al. (2019) designed and synthesized a new type of nanoparticle contrast agent, including self-assembled NaGdF₄ and CaCO₃ nanoconjugates, providing an integrated MRI nano platform with high tumor selectivity and good biocompatibility. In this design, the spatial confinement of the Gd³⁺ leads to an “OFF” MRI signal due to insufficient interaction between the protons and the crystal lattices. However, when immersed in the mildly acidic TME the embedded CCNM generate CO₂ bubbles and subsequently disconnect the nanoconjugates, thus resulting in an “ON” MRI signal. Due to the excellent performance of nanomaterials in vitro, Yi et al. (2019) investigated their comparative ability in vivo (Figure 5D). T1-weighted MR images were acquired in vivo before and after intravenous injection of nanoconjugates. The tumor site is relatively dark before injection of nanoconjugates, and it starts to light up about 30 min after injection. The contrast enhancement at the tumor site reached a tumor-to-background ratio of approximately 48 at 195 min postinjection. Compared with commonly used magnetic contrast agents, the in vivo performance of NaGdF₄-CaCO₃ shows a 60-fold enhancement in tumor visualization. It is suitable for deep tissue imaging, with higher sensitivity and selectivity, which is of great significance for constructing intelligent magnetic resonance imaging.

In addition, CCNM can generate CO₂ bubbles under acidic conditions, which can then enhance ultrasound imaging signals. Huang et al. (2020) developed a diagnostic nanoparticle system for ultrasound and fluorescence dual-mode imaging. The nanoparticle loaded with DOX showed the ability to simultaneously treat cancer. Therefore, loading DOX onto CCNM yields CaCO₃-DOX nanoparticles. Under acidic conditions, CCNM generate CO₂ to enhance ultrasound imaging and increase the release of DOX, thereby improving the quality of ultrasound imaging. Also, ICG was encapsulated into CCNM, to further detect the tumor with fluorescence. Overall, this study integrates imaging and therapeutic functions to provide a promising and attractive strategy for cancer treatment. Through research, it has been found that CCNM have certain help and advantages in the diagnosis of tumors. But there are still shortcomings such as instability and weak targeting of CCNM. It is necessary to improve their targeting and stability in order to maximize the diagnostic role of CCNM in tumors. Meanwhile, Vidallon et al. (2020) synthesized hybrid CCNM with biocompatibility and pH sensitivity through rapid precipitation method. Among them, CCNM can undergo decomposition under acidic conditions to generate high echo CO₂ bubbles, increasing the contrast intensity of ultrasound and demonstrating its potential as an ultrasound contrast agent.

Compared to traditional ultrasound contrast agents, CCNM not only achieve long-term stability of ultrasound imaging but also can quickly clear in the body without causing harm to the human body (Ma et al., 2022). At the same time, appropriate surface modification can also endow particles with specific targeting. The mesoporous structure generated by CCNM polymerization can encapsulate therapeutic drugs such as chemotherapy and sound sensitizers with good drug loading and release performance (Qiao et al., 2020). Therefore, combining the diagnosis and treatment functions of CCNM play an important role in improving the diagnosis and treatment technology of tumors.

The acid sensitivity of CCNM make them particularly attractive in cancer treatment because micro acidity is a characteristic of TME (Lu et al., 2021). CaCO₃ can be dissolved into non-toxic products (Ca²⁺, CO₃ ²⁻) in some acidic environments, which has been used as a drug delivery system (Zhao et al., 2022b; Chiang et al., 2022). Xu et al. (2022a) loaded anticancer drugs 5-fluorouracil (5-FU) and CUR with CCNM as carriers and placed the drug-loaded CCNM in phosphate buffers (pH 5.8, 6.6, 7.4) to observe the pH-dependent release of drugs. It was found that the release of 5-FU increased with the decrease of pH value, and the results showed that CCNM had good pH control drug release performance. Therefore, CCNM have great potential in acid responsive drug delivery.

The biosafety of nanoparticles is crucial in biomedical applications. The reduction in particle size and larger surface area makes them better absorbed by body fluids and tissues, thereby increasing their toxicity (Dong et al., 2022). Unlike other nanoparticles, CCNM have natural biocompatibility because Ca²⁺ and CO₃ ²⁻ ions are already widely present in the human body and have not shown cytotoxic effects in normal cell and in vivo studies (Yang et al., 2021b). Li et al. (2020b) observed through the use of rabbit red blood cells that CCNM did not cause any cell aggregation or dissolution, indicating that it has good blood compatibility.

The relatively large specific surface area of CCNM gives them high loading capacity. At the same time, the high porosity and well-developed internal structure of CCNM allow for the accommodation of molecules with different properties (Atchuda et al., 2022). These particles can effectively capture various bioactive substances, including low molecular weight compounds and large molecules. These bioactive substances are loaded onto CaCO₃ through physical adsorption into pores or co precipitation during particle formation, which is very important in delivery systems (Kanwal et al., 2022).

Due to high costs and potential material safety issues, new nano formulations used for targeted drug delivery are significantly hindered when transitioning from the laboratory to the clinical setting. CaCO₃ is the most abundant mineral in nature, its production cost is significantly lower than that of other nanoparticles, making it a cheap inorganic material (Kaiping et al., 2023). Therefore, CCNM are an extremely important material in the biomedical field. At the same time, compared with other nanomaterials, the preparation of CCNM only requires the use of basic salts (calcium salts and carbonates) and often does not require the use of organic solvents. This simple manufacturing process reduces the cost of CCNM (Trushina et al., 2022).

Easy functionalization is also one of the huge advantages of CCNM. Many studies have functionalized it through dry, wet, and in-situ modifications to enhance stability and targeting (Han et al., 2019). Due to its small particle size, high specific surface area, and high specific surface energy, CCNM are prone to particle aggregation and agglomeration during preparation and post-treatment, resulting in the formation of secondary particles, increasing particle size and losing the functionality of ultrafine particles during final use, thereby affecting practical application results (Lee et al., 2019; Ippolito et al., 2020). The key to solving this problem lies in the surface modification of CCNM, reducing the adhesion between particles, and improving its dispersibility and dispersion stability in the matrix (Li et al., 2019; Shen et al., 2019). Meanwhile, CCNM still cannot achieve precisely targeted therapy solely relying on passive targeting and acid responsiveness. Therefore, it is possible to endow the delivery system with targeting ability by coupling CCNM to targeted molecules. After the functionalization of CCNM, its in vivo stability in the blood will be higher, avoiding macrophage capture and possessing certain targeting characteristics, allowing the carrier to accumulate at specific sites (Dang et al., 2019). Li. et al. (2021d) developed a multifunctional CCNM for the targeted treatment of breast cancer and inhibition of metastasis. The CCNM were functionalized with folic acid molecules (FA) and coated with the T-cell membrane. Within the acidic TME, the CCNM undergo decomposition, leading to the release of Ca²⁺ ions and FA. The liberated FA molecules selectively bind to cancer cells via folate receptors and effectively suppress cancer cell migration, invasion, and proliferation. Notably, the incorporation of T cell membranes imparts tumor-targeting capabilities to the material while also preventing immune clearance within the body. This gives CCNM a prominent advantage in drug delivery systems for cancer treatment.

In normal individuals, there is also a certain amount of Ca²⁺, so it is difficult to achieve effective therapeutic effects. Moreover, the Ca²⁺ channel/pump on the cell membrane has a strong regulatory function, which makes the cell Ca²⁺ overload quickly return to the normal level, resulting in poor anti-cancer effects (Marchi et al., 2020; Tinawi, 2021). Therefore, combination therapy can further increase the Ca²⁺ content to achieve effective therapeutic concentration. In recent years, drug delivery systems constructed using CCNM biomaterials have been widely studied, but the concentration of drugs delivered is limited and cannot achieve clinically effective concentrations, which cannot achieve the desired clinical effects. Some studies have shown that various molecular adjuvants, such as biopolymers or synthetic polymers, can be added to the CCNM to load more drugs and make the drug release more slowly (Zhou et al., 2023).

Ca²⁺ is an element contained in the human body, which has good biocompatibility and biodegradability (Wang et al., 2021). However, when the Ca²⁺ content exceeds the limit, the effect of the Ca²⁺ steady-state regulation system is not very clear. Meanwhile, the inevitable leakage of Ca²⁺ in body fluids is a long-term issue for nanomedicine delivery systems (Bai et al., 2021). Excessive Ca²⁺ can trigger coagulation reactions, leading to the formation of blood clots and posing a threat to organisms (Wang et al., 2021). Moreover, a substantial number of studies solely assess the short-term toxicity of CCNM in mice by delineating organ damage subsequent to CCNM injection, which still poses challenges for accurately evaluating the biosafety of CCNM. So it is imperative to acknowledge the long-term potential risks associated with CCNM. Therefore, in order to facilitate the clinical translation of CCNM, it is imperative to conduct a comprehensive evaluation of the long-term effects of CCNM across various animal models, ranging from rodents to mammals.

CCNM have been widely studied in the field of tumor diagnosis and treatment, but there are still some challenges before clinical transformation (Dong et al., 2020). The present preparation processes of CCNM are instable, which easily leads to large particles (Vavaev et al., 2022). Hence, it is imperative to devise precise techniques enabling control over size, composition, and surface modification in order to facilitate large-scale production of CCNM. In addition, the prediction of drug release kinetics for CCNM remain challenging. Despite extensive research on the pH sensitivity of CCNM, their release behavior under normal physiological conditions has not been comprehensively evaluated.